Role of Tyrosine in Native Silk Fiber Spinning and its Application to Biomaterial Development

Native silk fibers exhibit strength and toughness values that rival those of the best synthetic fibers. In addition the silk worm is able to spin these fibers using only water as a solvent with fine control of applied shear, pH and salt concentrations to efficiently convert the spinning dope into a fiber. In contrast, synthetic polymers require harsh solvents and extreme processing conditions. These requirements result in the synthetic fibers being orders of magnitude more energy intensive to produce and generating chemical wastes that require disposal. It has been found that these fibers can be solubilized and utilized in the field of biomedical engineering as a biomaterial for tissue engineering and regenerative medicine applications. However, the processing of the silk fibroin into these scaffolds and matrices results in significant protein degradation. In order to provide material properties for the engineering of new materials, we examine how the regeneration process impacts the rheological and material properties of the resulting silk based biomaterials. Additionally, despite significant research on the spinning processes of silkworms and spiders, a comprehensive understanding of the mechanisms by which silkworms are capable of spinning such tough fibers eludes researchers. Here we propose that pi-pi interactions of the phenolic side chains on tyrosine residues provide a template to properly orient the silk molecules such that the crystalline domains are in registration and drives the self-assembly of the spinning dope. A combination of empirical and modeling based approaches elucidate the role of the tyrosine residues present in the semi-crystalline regions of the silk fibroin protein and how to exploit these interactions. The association of the tyrosine residues and correlation to self-assembly in solution was empirically determined by assessing the intrinsic fluorescence in combination with circular dichroism. In situ FTIR found that enzymatic crosslinking of the tyrosine residues initiated the immediate development of higher ordered combination. The degree of crosslinking was similarly found to correlate with the final crystallinity when the crosslinked samples were dehydrated. Molecular dynamic simulations were undertaken in order to understand the atomistic association of these protein residues. The findings are consistent with the empirical data suggesting that tyrosine is an important factor in the self-assembly of the silk proteins. The activity of the tyrosine residues and potential for crosslinking also provides for a facile method of generating materials with unique and tunable properties which will be explored and characterized. Thus, a greater understanding of the spinning process and role of the protein sequence has been determined and provided avenues for expanding the platform of silk biomaterials.